It is known that DNA damage causes DNA strand breaks, although it depends on the situation. The term “DNA strand breaks” refers to a state in which hydroxyl and phosphate groups that constitute DNA have dissociated. DNA double-strand breaks are caused by external factors such as radiation and internal factors such as DNA digestion. DNA damage is known to cause various reactions, including cell cycle arrest, DNA repair, and apoptosis (Non-Patent Document 1). All of these reactions are induced by ATR and ATM kinases belonging to the PIKK family that is important for controlling the DNA damage checkpoint mechanism and DNA repair (Non-Patent Document 2). Since DNA damage leads to destruction of well-organized mechanisms of cells, many mechanisms are thought to have evolved and to have been acquired for this phenomenon. Therefore, if it would become possible to detect DNA double-strand breaks in intact living cells, it would be useful for experimental reagents, test agents, and pharmaceutical compositions. In order to realize such detection, it must be able to easily detect changes in intact target cells. The ligand (NKG2D-L) of NKG2D belonging to the C-type lectin-like receptor family is known to be expressed on the surfaces of cells undergoing DNA breaks. This suggests that cells respond to DNA damage within a range that is greater than previously expected. In particular, DNA double-strand breaks correspond to damage that causes cleavage of both strands of DNA forming a double helix structure. It is most difficult for organisms such as cancer cells to repair the damage. However, when the repair system responds to the damage to repair it, the cells can survive. In most cases, such response occurs in the nucleus and then in the cytoplasm. Changes due to such response on the cell membrane have been little known. Under such circumstances, NKG2D-L is known as a protein that is expressed on the cell membrane in response to DNA damage. The expression of this protein is induced by ATR and ATM described above and then by CHK1 and CHK2 located downstream thereof, respectively (Non-Patent Document 3).
Profiling at the mRNA level using microarrays of X-ray-irradiated cells and non-irradiated cells revealed that the expression levels of various genes change due to DNA damage (Non-Patent Document 4). It is true that many candidates of DNA damage-responsive genes were found by this method. However, it was difficult to select marker molecules that can be actually applied in practice due to a disadvantage of mismatch of the mRNA expression level and the protein expression level seen in many aspects (Non-Patent Document 5). In order to solve these problems, comprehensive analysis was carried out by incorporating proteomics technology using two-dimensional electrophoresis. In this case, by taking into account that the abundance ratio of membrane proteins is usually significantly lower than that of intracellular proteins, comprehensive analysis was also carried out by incorporating a technology of selectively labeling and concentrating membrane proteins (Non-Patent Documents 6 to 8).
First, the present inventors created a method for obtaining an antibody against a protein expressed on the cell membrane (Patent Document 1 and Non-Patent Documents 9 and 10). In addition, the present inventors succeeded in the exhaustive acquisition of cell surface antigens and suggested high usefulness and potential of the method (Non-Patent Document 11).
As a DNA damage-responsive protein molecule, a phosphorylated histone protein molecule, called γ-H2AX, is known. Histones are a group of proteins that constitute a chromosome and play a role of folding DNA which is a very long molecule in the nucleus. H2AX is a member of the histone. One of the cellular responses induced when DNA double-strand breaks occur is that H2AX becomes phosphorylated on the 139 serine position. Phosphorylated H2AX is then called “γ-H2AX.” The use of a fluorescence-labeled antibody specific to γ-H2AX makes it possible to visually detect the sites of DNA double-strand breaks. γ-H2AX can be used in an environment in which reactions in the nucleus are detectable (Non-Patent Document 12).
Cancer is the leading cause of death in Japan. The number of cancer patients has been increasing each year with aging. The development of drugs and therapies with high efficacy and safety has been strongly desired. Conventional therapies such as chemotherapy and radiation are problematic because they can kill cancer cells, but at the same time, they cause damage to normal cells, resulting in induction of strong adverse reactions. To solve this problem, molecular target therapies are being actively studied, the therapies comprising designing a drug that targets a molecule specifically expressed in cancer cells and treating cancer with the drug. Among molecular targeted agents for cancer treatment, antibody drugs have been gaining a lot of attention because of their advantages, e.g., long half-life and fewer adverse reactions. Successful examples of the development of such agents include a chimeric antibody targeting CD20, called Rituxan, a humanized antibody targeting Her2/neu, called Herceptin, and a humanized antibody targeting the vascular endothelial growth factor (VEGF), called Avastin. These antibodies have been used for cancer as a target disease and the therapeutic effects have been recognized.
Antibodies used as therapeutic agents can be divided into labeled antibodies and unlabeled antibodies. It is believed that the mechanisms of unlabeled antibodies include the following: (1) antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in which immune system cells and molecules are involved; (2) inhibition of signals associated with intracellular survival and proliferation by target molecules; (3) induction of apoptosis; and (4) regulation of secretion of cytokine. Therapeutic effects can be exerted by causing the death of tumor cells or discontinuing the proliferation of tumor cells based on a combination of the above mechanisms. Labeled antibodies are obtained by linking antibodies to cytotoxic substances such as radioactive substances, toxins, enzymes, and drugs. By making use of the antibody specificity, labeled antibodies can be delivered only to cancer tissue, thereby improving the therapeutic effects and reducing the adverse reactions.
It is generally known that when a non-human animal antibody, e.g., a mouse antibody, is administered to a human, the antibody is recognized as a foreign substance so that a human antibody against the mouse antibody (Human Anti-Mouse Antibody; hereinafter referred to as “HAMA”) is induced in the human body. HAMA is known to react with a mouse antibody which is administered to a human body so as to cause adverse reactions (Non-Patent Documents 13 to 16), accelerate the disappearance of the mouse antibody from the human body (Non-Patent Documents 14, 17, and 18), and reduce the therapeutic effects of the mouse antibody (Non-Patent Documents 19 and 20).
Chimeric antibodies have been developed in order to avoid such problems (Patent Documents 2 and 3). A chimeric antibody has antibody regions derived from two or more species (e.g., a variable region of a mouse antibody and a constant region of a human antibody). Accordingly, chimeric antibodies are advantageous in that features of a mouse antibody are maintained while a human complement or cytotoxic activity can be stimulated because of the presence of human Fc. However, chimeric antibodies also induce the HACA (Human Anti-Chimera Antibody) response. Moreover, recombinant antibodies characterized in that only a substituted antibody part is a recombinant antibody complementarity-determining region (i.e., “CDR”) (Patent Documents 4 and 5). CDR transplant technology has been used to produce an antibody comprising a mouse CDR, a human variable region framework, and a human constant region, i.e., a “humanized antibody.” However, such humanized antibody is also immunogenic to humans and causes the HAHA (Human anti-Human Antibody) reaction (Non-Patent Documents 21 and 22). Therefore, in clinical application, more safe and effective antibody drugs having no immunogenicity have been awaited.
As an aside, it can be said that acquisition of an antibody capable of recognizing an “intact” target cancer antigen present on the cell membrane surface is essential for antibody drug discovery. However, since target cancer antigens are membrane proteins, it has been difficult to obtain antibodies even against known cancer antigens. In order to solve such problem, the present inventors have created a gigantic human antibody library consisting of as many as 100 billions of independent clones and established a method of exhaustive acquisition of antibodies against proteins (cell surface antigens) present on the cell membrane surfaces of cancer cells and tissues using the library (Patent Documents 6 to 8).